Endothelial Nitric Oxide Deficiency Reduces MMP-13–Mediated Cleavage of ICAM-1 in Vascular Endothelium

Abstract

Objective-Lack of endothelial nitric oxide synthase worsens atherosclerosis at least by increasing monocyte adhesion to endothelial cells. The purpose of this study was to elucidate the molecular mechanism elicited by NO. Methods and Results-We evaluated atherosclerosis in apoE and NOS3/apoE-deficient mice fed with high-cholesterol diet. We found significant increase in aortic lesion size, and infiltration of macrophages in NOS3/apoE-null mice when compared to apoE-deficient animals. To test the relevance of cellular adhesion as well as extracellular matrix degradation, we evaluated ICAM-1, VCAM-1, PECAM-1, MMP-2, MMP-9, MMP-12, MT1-MMP, and MMP-13 levels in mouse aortas. Lack of NO inhibits MMP-13 and increases ICAM-1 levels in atherosclerosis as compared to apoE-null mice. Ectopically expression of ICAM-1 in eukaryotic cells revealed that extracellular domain of ICAM-1 harbors a substrate recognized by MMP-13. Incubation of COS-7 cells expressing ectopic ICAM-1 in the presence of active MMP-13 induced inhibition of RAW 264.7 cell adhesion to COS-7 monolayers. MALDI-TOF MS analysis combined to Liquid chromatography coupled to Ion Trap MS on ICAM-1 incubated with MMP-13 allowed us to determine the cleavage sites of MMP-13 at positions E61 and G98 of ICAM-1. G98 is part of a PDGQS moiety, which shows homology with the consensus PDGLS substrate located at the MMP-13 cleaved site of type II collagen I-alpha. (ICAM-1). In aortas of apoE-null mice, ICAM-1 was found located in lesion-prone sites of the aorta, 2 and the importance of this inflammatory molecule was evidenced in atherosclerotic mice deficient in ICAM-1, which resulted protected from atherosclerotic lesions. 3 The role of matrix metalloproteinases (MMPs) has been analyzed in detail in late atherosclerosis. 4,5 However, the precise role of MMPs in the early steps of this pathology is still debated. In this regard, the effect of MMPs in the shedding of adhesion molecules was investigated in vitro, 6 although no data were reported in the context of atherosclerosis. Conclusions-TakingLack of endothelial NOS (eNOS, NOS3) increases leukocyte-endothelial adhesion, smooth muscle cell migration, platelet aggregation, and atherosclerosis in mice. 7,8 However, the mechanisms by which NOS3 could prevent atherosclerosis still remain unclear. Here we found that in atherosclerotic NOS3/apoE-deficient mice, increased monocyte adhesion, and a significant reduction of MMP-13 expression, were detected when compared to apoE-deficient animals. In addition, we found that MMP-13 cleaves ICAM-1 both in vivo and in vitro. Mass spectrometry analysis revealed 2 sites at positions E61 and G98, close to the ICAM-1 extracellular N-terminal domain. The relevance of MMP-13 and ICAM-1 on cellular adhesion was found in COS-7 cells expressing ectopic ICAM-1, in which RAW 264.7 cell adhesion was inhibited by the presence of active MMP-13. Our findings may help to explain at the molecular level the protective effect of endothelial NO in atherosclerosis. Methods ReagentsGeneral cell culture ...

The expression of matrix metalloproteinase-13 is increased in vessels with blood–brain barrier impairment in a stroke-prone hypertensive model

“…In this study, the immunostaining for MMP-13 was not clear in the endothelial cells of vessels. But, the role of MMP-13 in the endothelial cells cannot be disregarded because MMP-13 in endothelial cells is known to play a role in protection of atherosclerosis, 30 which may affect barrier function. Nagel et al 32 noted that two processed forms (47 and 20 kDa) of MMP-13 appeared in the brain after ischemia.…”

“…To quantify gene expression, TaqMan real-time quantitative PCR was performed with the ABI PRISM 7700 Sequence Detection System using Assays on-Demand Gene Expression probes of MMPs (MMP-2, MMP-3, MMP-9 and MMP-13), or glyceraldehydes 3-phosphate dehydrogenase (GAPDH) and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), as reported earlier. 30 The PCR cycling conditions for all samples were as follows: 501C, 2 min for AmpErase UNG activation; 951C, 10 min for AmpliTaq Gold activation; and 50 cycles for melting (951C, 15 s) and annealing/extension (601C, 1 min) steps. 16,31 Each sample was run in duplicate, and each PCR experiment included two nontemplate control wells.…”

Adiponectin resistance and vascular dysfunction in the hyperlipidemic state

“…When considering where endothelial dysfunction in hyperlipidemia might originate, among the most likely mechanisms is the decreased synthesis of bioactivated NO. Besides its vasodilatory effects, NO has many reported antiatherogenic properties, including reducing platelet aggregability [40] , limiting vascular smooth muscle cell proliferation [41] , inhibiting adhesion molecule expression in endothelium [42] , inhibiting neutrophil and monocyte adhesion to the endothelium [43,44] , and preventing monocyte chemotaxis [45] . Chronic provision of L-arginine to the diets of hypercholesterolemic rabbits has been reported to improve endothelium-dependent vasodilation and reduce the extent of atherosclerotic lesions [46] .…”

ICAM-1: Contribution to Vascular Inflammation and Early Atherosclerosis

“…The inflammatory cytokines TNF-, IL-1 and IFN-have been reported to induce shedding of sICAM-1 from the cell surface of different primary cells and cell lines (Becker, Dummer et al 1991;Leeuwenberg, Smeets et al 1992;Pigott, Dillon et al 1992;Fonsatti, Altomonte et al 1997;Leung 1999). Furthermore, several proteases including matrix metalloproteases (MMP) (Lyons and Benveniste 1998); Tarín, Gomez, et al 2009), human leukocyte elastase (Champagne, Tremblay et al 1998) and TNF- converting enzyme (TACE) (Tsakadze, Sithu et al 2006) have been demonstrated to cleave sICAM-1 from the cell surface. The cleavage site on ICAM-1 seems to vary depending on cell type, glycosylation pattern and therefore also on the proteases involved.…”

“…The cleavage site on ICAM-1 seems to vary depending on cell type, glycosylation pattern and therefore also on the proteases involved. Further analysis showed that cleavage of membrane bound ICAM-1 by MMP-13 is dependent on NO and might be part of NO-media ted atheroprotection (Tarín, Gomez et al 2009). …”